Carbohydrates are vital in nature, not only for energy metabolism, but also as structural scaffolds, recognition motifs, solubility aids, and functional modulators. Yet despite the vast structural and functional diversity of natural glycoconjugates, they are constructed via common biosynthetic themes. Specifically, sugars are attached to most proteins, lipids, carbohydrates, and small molecules by glycosyltransferases which, with few exceptions, use sugar nucleotides as the monosaccharide donors. These sugar nucleotides are constructed from sugar-1-phosphates and NTPs by sugar-1-phosphate nucleotidyltransferases, also referred to as sugar nucleotide pyrophosphorylases (E.C. 2.7.7.-), providing the precursors (usually ADP-, CDP-, GDP-, UDP- and dTDP-glucoses, as well as GDP-mannose, GDP-fucose, and UDP-N-acetylglucosamine) central to nearly all glycosylation-dependent processes.
Nucleotidyltransferases are prevalent in nature [there are currently ˜14,000 known and putative nucleotidyltransferase sequences in GenBank], are often allosterically controlled, and generally proceed via ordered bi-bi mechanisms. For example, the forward reaction catalyzed by Salmonella glucose-1-phosphate thymidylyltransferase (RmlA), set forth herein as SEQ ID NO:1 (deposited as GenBank Accession No. CAA40117), proceeds via direct SN2 attack upon the NTP α-phosphate by an α-D-sugar anomeric phosphate to provide the desired sugar nucleotide and pyrophosphate (FIG. 1). Nucleotidyltransferases from both prokaryotes and eukaryotes have reported flexibility toward variant sugar phosphates in vitro and the uniquely broad sugar-1-phosphate tolerance of RmlA has been exploited for the synthesis of diverse UDP- and dTDP-based sugar nucleotide libraries and enhanced via structure-based engineering. To date, more than 30 different sugar-1-phosphates have been reported as substrates for RmlA variants.
The corresponding pyrimidine-based sugar nucleotide libraries have served as the foundation for a process known as natural product glycorandomization (FIG. 1B)—an enzymatic strategy to exchange natural product sugars with diverse sugar arrays. To date this strategy has been applied toward the diversification of glycopeptide, coumarin and macrolide antibiotics, as well as anthelmintic avermectin and enediyne anticancer agents. Yet while there exist limited reports wherein sugar-1-phosphate guanylyl- or adenylyl-transferases displayed moderate sugar-1-phosphate flexibility, the lack of a general strategy to generate diverse purine-based sugar nucleotide libraries excludes the corresponding diversification of many natural products glycosylated by purine sugar nucleotide-dependent glycosyltransferases.
Recently, thermophilic uridylyl- and thymidylyl-transferases were revealed to utilize alternative nucleotides, including both ribo- and deoxyribo-variants of one or more purine nucleotides. However, no single enzyme has been reported to utilize all eight naturally occurring NTPs. As can be appreciated, a single “universal” nucleotidyltransferase is highly desirable and would greatly benefit the production of diverse chemical libraries enzymatic and, in particular, enzymatic glycorandomization methodology.